Background: Recent evidence suggests that PD-L1 expression can be induced with radiotherapy and may be a mechanism for resistance to radiotherapy and immunotherapy. Sequentially assessing PD-L1 expression on cancer associated cells in circulation during treatment regimens may be a way to assess the efficacy of radiotherapy and immunotherapy in clinical trials. For this feasibility study, we evaluated the association of RAD50 induction, and PD-L1 expression, on CTCs and Cancer Associated Macrophage-Like Cells (CAMLs) in lung cancer patients (pts) before and during radiotherapy to determine expression changes of these markers.

Methods: Eleven pts with stage I-IV lung cancer were included in this pilot study. Three pts received Stereotactic Body Radiation Therapy (SBRT) for stage I disease and 8 other pts received chemoradiation for stage II-IV disease. Baseline blood samples (7.5 ml) were drawn prior to the start of radiotherapy (T0) and a second blood sample was drawn at a follow up visit during radiotherapy; or for three pts, after completing SBRT (T1); for a total of 22 samples. Blood was processed using CellSieve™ microfiltration (Creatv Microtech), stained for cytokeratin 8, 18 & 19 and CD45, and imaged. Using the QUAS-R (Quench, Underivatize, Amine-Strip and Restain) technique to remove fluoresce signal, all cells were restained for RAD50-AlexaFluor550 and PD-L1-AlexaFluor 488, along with DAPI nuclear stain. The RAD50 foci numbers within nuclear regions were quantified. PD-L1 pixel intensity was measured by the ZenBlue software and grouped into 4 IHC groups: 0-negative (pixel average 0-215), 1-low (pixel average 216-300), 2-medium (pixel average 301-750), and 3-high (pixel average 751+).

Results: There was at least one cytokeratin positive cell (i.e. CTC or CAMLs) found in each of the samples. Specifically CTCs were found in 82% of T0 and 64% of T1 samples, and CAMLs were found in 91% of T0 and 100% of T1 samples. RAD50 foci ranged from 0-16 per cell, with an average of 0.69 at T0 that increased to 3.46 at T1 (p=0.002) during radiotherapy. Distinctively, there were 6 pts with greater than 2 fold RAD50 foci increase at T1 and 5 pts with ≤ 2 fold induction. PD-L1 expression ranged from 34-2004 pixel intensity, with an average of 170 at T0 and 336 at T1 (p=0.08). Interestingly, 4 pts had no PD-L1 expression at T0 but an increase to 2 to 3+ at T1, 4 pts with low/no PD-L1 expression remained low at T1, and 3 pts had high PD-L1 expression that remained high or decreased at T1. There was no correlation between RAD50 induction and PD-L1 expression.

Conclusion: Both RAD50 foci and PD-L1 expression were quantifiable in both CTCs and CAMLs, and had variable responses to radiotherapy +/- chemotherapy. These data suggest that sequential tracking of CTCs or immune-related cells from the primary lung tumor is feasible using microfiltration and potentially can serve as predictive biomarkers for cancer therapy.

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